DESCRIPTION: Human pluripotent stem cells (hPSCs) provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating human somatic cells and tissues for developmental studies, toxicity testing, and cellular therapies. hPSCs are a particularly promising source of cardiac tissues since cardiomyocytes (CMs) cannot easily be attained from primary sources and are of tremendous importance in disease and pharmaceutical evaluation. In the current project period we developed an efficient, completely defined protocol to produce human CMs from hPSCs via temporal modulation of canonical Wnt signaling using small molecules, solving one of the major challenges in cardiac applications of hPSCs. However, current CM differentiation protocols fail to achieve mature cells possessing adult-like phenotypes, limiting the potential of these hPSC-derived CMs in regenerative applications. In prior work we also found that a different temporal modulation of Wnt signaling directs hPSCs to vascular endothelial cells (ECs). During heart development, paracrine interactions between developing ECs and CMs guide formation of cardiac tissue structure and function, but these signals are not present in current CM differentiation platforms. Thus, in the proposed project we will develop novel 2D and 3D platforms to co-differentiate hPSCs to spatially patterned ECs and CMs and use these platforms to test the hypothesis that paracrine interactions during EC-CM co- differentiation enhance CM maturation. We propose to use our team's expertise in biomaterials, stem cell biology, cardiac cell biology, genome editing, and cardiovascular development to extend our advances on differentiating pure populations of CMs and ECs from hPSCs via temporal modulation of Wnt signaling to include spatial regulation of Wnt signaling, enabling co-differentiation of CMs and ECs in precisely-designed patterns. In addition, we will use co-differentiation platforms to identify EC-generated paracrine mechanisms mediating CM maturation. Our specific aims to test the hypothesis of this application are: 1. Employ optogenetic spatial and temporal regulation of Wnt signaling to pattern hPSC co- differentiation to ECs and CMs in 2D. Co-differentiation will be enabled by light-mediated spatio-temporal activation and repression of Wnt signaling. 2. Implement release of Wnt modulators to pattern hPSC co-differentiation to ECs and CMs in 3D. Co- differentiation will be enabled by localized release of Wnt inhibitors in differentiating 3D hPSC aggregates. 3. Elucidate mechanisms of EC signaling to CMs during co-differentiation. Biochemical and genetic mechanisms will be employed to identify paracrine signaling that stimulates CM maturation in the 2D and 3D co-differentiation platforms developed in Aims 1 and 2.